The objective of this study is to determine the molecular determinants of synergistic inhibition of the Hepatitis C virus (HCV) polymerase when multiple allosteric inhibitors bind simultaneously to the enzyme. HCV affects close to 200 million people worldwide, making it a global health concern. The HCV polymerase (gene product NS5B) is a vital component of viral replication and has no known mammalian homolog, making it a promising target for antiviral therapeutics. Unfortunately, the low fidelity of NS5B results i quasi-species of the virus, leading to multiple enzyme variants and making it difficult to target the HCV NS5B with single inhibitors. Recent studies show that the use of multiple allosteric inhibitors has a synergistic inhibitory effect on NS5B. However, the mechanism by which this synergistic inhibition is unclear. Here, we propose to employ computational and experimental approaches to determine the changes in both dynamic and thermodynamic properties that are critical for mediation of the synergistic inhibition of NS5B. Such approaches will provide us with information on how inhibitor-binding impacts the motions of protein residues and the enzyme free energy landscape resulting to induce synergistic inhibition of NS5B. Furthermore, mechanistic details revealed by our study on NS5B may be relevant to the areas of drug discovery, regulation of metabolic pathways, and other signal transduction processes.